Hydrolysis of O-Acetyl-ADP-ribose Isomers by ADP-ribosylhydrolase 3

Abstract

O-Acetyl-ADP-ribose (OAADPr), produced by the Sir2-catalyzed NAD ؉ -dependent histone/protein deacetylase reaction, regulates diverse biological processes. Interconversion between two OAADPr isomers with acetyl attached to the C-2؆ and C-3؆ hydroxyl of ADP-ribose (ADPr) is rapid. We reported earlier that ADP-ribosylhydrolase 3 (ARH3), one of three ARH proteins sharing structural similarities, hydrolyzed OAADPr to ADPr and acetate, and poly(ADPr) to ADPr monomers. ARH1 also hydrolyzed OAADPr and poly(ADPr) as well as ADP-ribose-arginine, with arginine in ␣-anomeric linkage to C-1؆ of ADP-ribose. Because both ARH3-and ARH1-catalyzed reactions involve nucleophilic attacks at the C-1؆ position, it was perplexing that the ARH3 catalytic site would cleave OAADPr at either the 2؆-or 3؆-position, and we postulated the existence of a third isomer, 1؆-OAADPr, in equilibrium with 2؆-and 3؆-isomers. A third isomer, consistent with 1؆-OAADPr, was identified at pH 9.0. Further, ARH3 OAADPr hydrolase activity was greater at pH 9.0 than at neutral pH where 3؆-OAADPr predominated. Consistent with our hypothesis, IC 50 values for ARH3 inhibition by 2؆-and 3؆-N-acetyl-ADPr analogs of OAADPr were significantly higher than that for ADPr. ARH1 also hydrolyzed OAADPr more rapidly at alkaline pH, but cleavage of ADP-ribose-arginine was faster at neutral pH than pH 9.0. ARH3-catalyzed hydrolysis of OAADPr in H 218 O resulted in incorporation of one 18 O into ADP-ribose by mass spectrometric analysis, consistent with cleavage at the C-1؆ position. Together, these data suggest that ARH family members, ARH1 and ARH3, catalyze hydrolysis of the 1؆-O linkage in their structurally diverse substrates.Mono-ADP-ribosylation is a post-translational modification, in which the ADP-ribose (ADPr) 5 moiety of NAD is transferred to an acceptor protein (1). This modification serves as the mechanism by which several bacterial toxins (e.g. Pseudomonas exoenzyme S, cholera toxin, diphtheria toxin) exert their effects on mammalian cells (2, 3). Mammalian cells also produce endogenous ADP-ribosyltransferases that catalyze reactions similar to the bacterial toxins, specifically, the ADPribosylation of arginine residues in proteins (4). In addition, mammalian cells possess hydrolases that cleave the ADPr-protein linkage, releasing ADPr and regenerating the unmodified protein (5, 6). An ADP-ribosyl(arginine) hydrolase, termed ARH1, catalyzes in a stereospecific manner, hydrolysis of the ␣-linkage of arginine-ribose found in ADP-ribosyl(arginine)-protein to ADPr and (arginine)-protein (7, 8), consistent with the regulation of ADP-ribosyl(arginine)-protein levels by opposing activities of transferases and hydrolases, participating in an ADP-ribosylation cycle (4, 9).Three known members (ARH1-3) of the ARH family of proteins are similar in molecular size (ϳ39 kDa) and amino acid sequence (10). As noted above, ARH1 catalyzes the hydrolysis of ADP-ribose-arginine and also hydrolyzes ADP-ribose linkages to guanidine. The reaction is stereospecific, and only the ␣-ano...

Structure and function of the ARH family of ADP-ribosyl-acceptor hydrolases

“…ARH1 and ARH3 have different substrate specificities. ARH1 cleaves mono-ADP-ribosylated substrate with the modification on an arginine, while ARH3 hydrolyzes PAR and O AADPr [76, 78, 81, 82]. ARH2 has not been shown to have enzymatic activities related to mono- and poly-ADP-ribose [81, 82].…”

“…ARH1 and ARH3 preferentially cleave the α-anomer at the C-1″ position of ADP-ribose attached to arginine and ADP-ribose in PAR, respectively. ARH3 hydrolyzes O AADPr in the same manner [81, 82]. Similar to ARH3, ARH1 cleaves PAR and O AADPr, but ARH1 activity is less than 1% that of ARH3, indicating that ARH1 might possess weak hydrolytic activity toward the O -glycosidic bond [81].…”

“…On the other hand, ADP-ribosylated cysteine, asparagine, and diphthamide, synthesized by bacterial toxins, are not substrates hydrolyzed by ARH1 or ARH3 [76]. ARH1 and ARH3 activities are competitively inhibited by ADP-ribose, but not ribose 5-phosphate, AMP, ADP or NAD + [80–82]. Thus, ARH1 and ARH3 recognize the ADP-ribose moiety of the substrate.…”

“…Thus, ARH1 and ARH3 recognize the ADP-ribose moiety of the substrate. Although ARH2 binds ADP-ribose, it has not been shown to be enzymatically active [81, 82]. …”

Functional Role of ADP-Ribosyl-Acceptor Hydrolase 3 in poly(ADPRibose) Polymerase-1 Response to Oxidative Stress

“…Although ARH3 shows significant structural similarities to ARH1, substrates of ARH3 differ from those of ARH1. To date, ARH3 has been identified as hydrolyzing poly(ADP-ribose) (PAR) and O -acetyl-ADP-ribose ( O AADPr), an NAD + -dependent sirtuin reaction product, but not mono-ADP-ribosylated arginine, the primary substrate of ARH1 [10–13]. ARH2 has not been reported to have any enzymatic activity, because of the substitution of amino acids critical for enzymatic activity, which are conserved in both ARH1 and ARH3 [11, 12].…”